1. Field of the Invention
The present invention relates generally to the field of mammalian cell biology. More particularly, it concerns methods and compositions using apoptosis inhibitors to increase the efficiency of nuclear transfer, useful in the production of transgenic and non-transgenic mammals from cultured cells or cell lines. Methods using apoptosis inhibitors in cloning mammals, and for producing chimeric cell lines, transgenic and chimeric mammalian tissues and mammals are also provided.
2. Description of Related Art
The basic procedure for nuclear transfer concerns obtaining single cells and fusing them to enucleated recipient ovum. This effectively transfers the nucleus of the donor cell into the recipient cytoplasm where, if successful, it is reprogrammed and subsequently instructs development of a new embryo that is genetically identical to that from which the cell was acquired. Nuclei from embryonic fibroblasts as well as adult mammary epithelial cells can direct normal development in the sheep (Wilmut et al., 1997).
Although the nuclear transfer technique is less advanced in pigs, there have been reports of successful births using nuclei from 4-cell embryos (Prather et al., 1989). Primordial germ cells (PGCs) collected from fetal tissue have also been successfully utilized as donors for nuclear transplantation (Cherny and Merei, 1994, Delhaise et al., 1995, Lavoir et al., 1997, Strelchenko, 1996). In pigs it has been demonstrated that previously cryopreserved PGCs can be used successfully as nuclear donors, giving rise to nuclear reprogramming and cleavage to the 4-cell stage (Liu et al., 1995). Additionally, nuclear reprogramming in cultured ICM-derived pig cells after nuclear transfer has been reported (Ouhibi et al., 1996). The ability of the embryos to participate in normal development was not studied.
In a recent study in cattle, 9-13% of cleaved nuclear transplant embryos developed to the blastocyst stage when oogonia collected from female fetuses (50-70 days gestation) was utilized as nuclei donors (Lavoir et al., 1997). Although no live calves were produced, an abnormal conceptus developed in one animal that had received 4 embryos. This conceptus was recovered by induced abortion at day 43 after failing to detect a heartbeat, and genetic analysis showed the fetus to be genetically identical to the donor oogonia. Similar results using bovine PGCs from both male and female fetuses have been reported (Moens et al., 1996). The observation that nuclei from cultured bovine PGCs can direct development up to day 60 with no significant fetal abnormalities reported suggests that, when PGCs are placed in culture, nuclear changes occur that increase the nuclear potency of the cells when compared with freshly isolated PGCs (Strelchenko, 1996).
In spite of the foregoing reports, the technique of nuclear transfer is plagued by extremely low efficiency. Thus, methods and compositions that increase the efficiency of nuclear transfer, using both cultured somatic and germ cells, would represent a significant advance in the art.
The present invention overcomes one or more of the shortcomings in the art by providing methods and compositions using apoptosis inhibitors to increase the number of cells available for manipulation, including homologous recombination and gene targeting, in the generation of cell lines, transgenic and chimeric tissues and animals. The methods and compositions of the invention increase the efficiency of nuclear transfer, for use in the production of transgenic and non-transgenic mammals from cultured cells or cell lines, in cloning mammals, and for producing chimeric cell lines, transgenic and chimeric mammalian tissues and mammals.
The present invention thus provides methods and compositions for increasing, and preferably significantly increasing, the number and/or proportion of nuclear transfer-competent cells within a mammalian cell population. These methods and compositions comprise contacting, providing, administering, admixing or culturing a mammalian cell population with an amount of at least a first apoptosis inhibitor effective to increase, and preferably significantly increase, the number and/or proportion of nuclear transfer competent cells within the mammalian cell population.
The invention further provides methods and compositions for performing nuclear transfer in which the efficiency of nuclear transfer is increased, and preferably, significantly increased. Such methods and compositions comprise maintaining or culturing a mammalian cell population in the presence of an amount of at least a first apoptosis inhibitor effective to increase, and preferably significantly increase, the number and/or proportion of nuclear transfer competent cells within the cell population and fusing at least a first nuclear transfer-competent cell therefrom with a suitable enucleated, recipient cell or ovum.
A xe2x80x9cnuclear transfer-competent cellxe2x80x9d, as used herein, means a cell capable of being used in conjunction with a suitable enucleated, recipient cell in an effective nuclear transfer method. The present invention therefore provides methods and compositions for increasing, and preferably significantly increasing, the number and/or proportion of nuclear transfer-competent cells from within mammalian cell populations contemplated for use in nuclear transfer.
xe2x80x9cNuclear transfer-competent cellsxe2x80x9d are preferably in the G0/G1 stage of the cell cycle and, as used herein, are xe2x80x9cviablexe2x80x9d in the sense that they are capable of effectively participating in nuclear transfer in conjunction with suitable enucleated, recipient cell(s) to produce functional nucleated cells, reprogrammed nucleated cells, and reprogrammed nucleated cells capable of instructing the development of a new embryo. The invention thus provides methods and compositions for increasing, and preferably significantly increasing, the number and/or proportion of viable, nuclear transfer-competent cells (nuclear transfer-compeient, viable cells) from within cell populations contemplated for use in nuclear transfer.
In such methods and compositions of the invention, it is the contact, provision, administration, admixture or culture of the cell population comprising the cells for use in nuclear transfer in the presence of an effective amount of at least a first apoptosis inhibitor that increases or significantly increases the number and/or proportion of viable, nuclear transfer-competent cells.
To complete the nuclear transfer process, the viable, nuclear transfer-competent cell or cells is/are fused with suitable enucleated, recipient cell(s), thereby achieving nuclear transfer, i.e., transfer of the donor nucleus into the enucleated cell to produce a viable, nucleated cell, reprogrammed nucleated cell and/or reprogrammed, nucleated cell capable of instructing the development of a new embryo.
Accordingly, the invention provides increasingly effective and efficient methods and compositions for performing nuclear transfer. Such methods and compositions comprise culturing or maintaining a mammalian cell population containing at least some cells or a sub-population of cells at the G0/G1 stage of the cell cycle in media comprising an effective amount of at least a first apoptosis inhibitor, thereby increasing the number and/or proportion of viable cells within the G0/G1 stage cells of said cell population; and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum. The xe2x80x9ceffective amountxe2x80x9d of the at least a first apoptosis inhibitor is an amount effective to increase the number and/or proportion of viable G0/G1 cells in the G0/G1 sub-population or overall cell population.
In the methods and compositions for performing nuclear transfer of the invention, the xe2x80x9cmammalian cell populationxe2x80x9d is xe2x80x9cat least a first mammalian cell populationxe2x80x9d, which at least a first mammalian cell population comprises at least one, some or a sub-population of cells at the G0/G1 stage of the cell cycle. Culture or maintenance with an effective amount of at least a first apoptosis inhibitor thereby increases the number and/or proportion of viable cells within the G0/G1 cells of said sub-population or said overall cell population.
In certain embodiments, the invention provides methods of performing nuclear transfer that comprise culturing at least a first mammalian cell in serum starvation media comprising at least a first apoptosis inhibitor for a period of time effective to arrest the at least a first cell at the G0/G1 stage of the cell cycle, and fusing the cell cycle arrested cell with an enucleated mammalian ovum.
Any method may be employed to initially obtain a mammalian cell population comprising potentially nuclear transfer competent cells, preferably a mammalian cell population comprising potentially viable G0/G1 cells, such that execution of the invention increases the actual nuclear transfer competent cells or actual viable G0/G1 cells within the cell population. Suitable methods that induce cells within a cell population to enter the G0/G1 stage of the cell cycle include those involving chemical treatment, nutrient deprivation, growth inhibition, manipulation of gene expression or combinations thereof. A preferred method of the invention is to culture the cell population in serum starvation media.
In the methods and compositions of the invention, it is the contact, provision, administration, admixture or culture of the cell population in the presence of an effective amount of at least a first apoptosis inhibitor that increases or significantly increases the number and/or proportion of viable, nuclear transfer-competent cells. The xe2x80x9ceffective amountxe2x80x9d of at least a first apoptosis inhibitor refers to both an effective mass and concentration, and to an effective period of time that the cell population is exposed to the apoptosis inhibitor.
The interplay of the xe2x80x9ceffective amounts and timesxe2x80x9d will be known those of ordinary skill in the art in light of the present disclosure. By way of example, it will be understood that, within the teachings of the present disclosure, xe2x80x9ceffectivexe2x80x9d contact with apoptosis inhibitors overall can be achieved using a lower amount or concentration for a longer time, or a higher amount or concentration for a shorter time.
To the extent that, in certain aspects of the present invention, there may be an xe2x80x9cinterdependencexe2x80x9d of time and dosage, the determination of xe2x80x9ceffective amountsxe2x80x9d is still within the level of skill in the art in light of the present disclosure. For example, in reference to Example I, the ordinary skilled artisan will understand that the use of MAC is preferred at mid-levels of the disclosed ranges, whereas NAC is preferred at high levels of the disclosed ranges and above. The ordinary skilled artisan will also understand that maximal effects result when MAC is present substantially throughout the culture or xe2x80x9cincubation periodxe2x80x9d of the cell population, whereas NAC need only be present at, substantially at, or at a time proximal to, the initial stages of the culture or incubation to have its most beneficial effect.
In certain embodiments, the mammalian cell population will contain somatic or germ cells from a mammal, whether immature or adult, a fetus or an embryo, such that the invention provides viable somatic or germ cells from a mammal in G0/G1. Preferred somatic cells include, but are not limited to, mammary gland cells and granulosa cells. Preferred germ cells include, but are not limited to, primordial germ cells (PGCs), fetal lung fibroblast cells and embryonic fibroblast cells, for example bovine or porcine embryonic fibroblast cells. The methods and compositions of the present invention may be used in conjunction with those of co-owned, co-pending U.S. application Ser. No. 08/949,155, filed Oct. 10, 1997, specifically incorporated herein by reference.
Accordingly, as disclosed in U.S. application Ser. No. 08/949,155, incorporated herein by reference, the present invention further pertains to methods of growing or culturing cells, preferably fetal or embryonic fibroblasts or primordial germ cells, comprising growing a cell culture or population comprising the cells of interest, preferably fetal or embryonic fibroblasts or primordial germ cells, on an effective density of feeder cells and in a biologically effective culture medium comprising an amount of at least a first apoptosis inhibitor effective to increase the number of nuclear transfer competent cells when said cell culture or population is grown, cultured or maintained under conditions and for a time sufficient to obtain undifferentiated cells, preferably undifferentiated fetal or embryonic fibroblasts or primordial germ cells, including nuclear transfer competent cells. The use of bovine and porcine cells in such methods is currently preferred.
U.S. application Ser. No. 08/949,155, incorporated herein by reference, exemplifies various effective feeder cells and effective densities thereof, as well as various biologically effective culture media. All such feeder cells, media components and concentrations may be used in the present invention. The cells and media in U.S. application Ser. No. 08/949,155 are exemplary only, any many such feeder cells, densities and biologically effective culture media are known and can be used in conjunction with the present invention.
The methods and compositions of the invention include those wherein the cell population comprises viable G0/G1 cells that comprise at least a first exogenous DNA segment. In that the present invention increases the efficiency of homologous recombination, gene targeting and nuclear transfer, viable G0/G1 cells that comprise at least a first exogenous DNA segment, and resultant cells, cell lines, blastocysts, oocytes, embryos and animals, are advantageous aspects of the invention.
As disclosed in U.S. application Ser. No. 08/949,155, incorporated herein by reference, the variety of exogenous DNA segments that may be included with the present invention is virtually limitless. The selected DNA segment may comprise at least a first coding region encoding a selected protein. However, protein production is not a requirement of the invention, which may be effectively practiced, e.g., using antisense or ribozyme technology, wherein the expression of an RNA species provides a desired phenoty sult. Where protein expression is desired, an exogenous coding region may encode a selected marker protein, such as green fluorescent protein (GFP).
In important embodiments, an exogenous coding region will be supplied that encodes an RNA or protein with a desired biological activity, including proteins that are physiologically or pharmacologically active (or rendered physiologically or pharmacologically active upon expression). In preferred embodiments, the encoded protein confers disease resistance, carcass composition, weight gain, coat composition or is a milk component protein.
An encoded RNA or protein may be physiologically or pharmacologically active only in specific tissues or may be active in a variety of sites or tissues. Proteins that are converted to an active form in an animal, e.g., through the action of enzyme-assisted transformation, pH, specific organ activities, and such like, or through the application of at least one more exogenous agent(s) are included. Proteins may also be adapted to increase expression in the chosen animal, e.g., by altering the coding sequence of the protein to use codons that are preferred for use in the particular animal.
Suitable examples of encoded products for use with the present invention include transcription or elongation factors, cell cycle control proteins, enzymes, kinases, phosphatases, DNA repair proteins, oncogenes, tumor suppressors, cytotoxins, angiogenic proteins, anti-angiogenic proteins, apoptosis-inducing agents, anti-apoptosis agents, immune system proteins, antigens, immune response stimulating proteins, cell surface receptors, accessory signaling molecules, transport proteins, enzymes, anti-bacterial, anti-microbial, anti-parasitic or anti-viral proteins or polypeptides.
Further suitable examples include hormones, neurotransmitters, growth factors, growth factor receptors, hormone receptors, neurotransmitter receptors, adhesion ligands, binding proteins, interferons, interleukins, chemokines, cytokines, colony stimulating factors and chemotactic factor proteins. Yet further examples are extracellular matrix components, molecules, ligands and peptides, such as collagens, fibrin, fibronectin, vitronectin, hyaluronic acid, RGD-containing peptides or polypeptides. Even further examples are blood proteins and muscle proteins and components.
Certain particular examples, as disclosed in U.S. application Ser. No. 08/949,155, specifically incorporated herein by reference, are SREHP, GP63, actinobacillus, pleuropneumoniae, pseudomonas aeruynosa, OprF, myelin basic protein, insulin, hCD59, DAF (CD55), factor IX, urokinase, xcex1-antitrypsin, tissue plasminogen activator, protein C, activin, adenosine deaminase, angiotensinogen I, antithrombin III, alpha I antitrypsin, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein E, atrial natriuretic factor, chorionic gonadotropin, alpha chain, beta chain, pro (rennin) chymosin, factor B complement, complement C2, complement C3, complement C4, complement C9, corticotropin releasing factor, epidermal growth factor, c-erb B, epoxide dehydratase, erythropoietin, C1 esterase inhibitor, factor VIII, factor IX, Christmas factor, factor X, fibrinogen A alpha, gamma B beta, gastrin releasing peptide, prepro glucagon, growth hormone, RF growth hormone, somatocrinin, hemopexin, inhibin, prepro insulin, insulin-like growth factor I, insulin-like growth factor II, alpha interferon, multiple leukocyte, fibroblast beta interferon, gamma interferon, interleukin-1, T-cell interleukin-2, growth factor, interleukin-3, two forms kininogen, beta subunit leuteinizing hormone, leuteinizing hormone, releasing hormone, lymphotoxin, mast cell growth factor, beta subunit nerve growth factor, PGDF c-sis oncogene, chain A, pancreatic polypeptide, icosapeptide, parathyroid hormone, prepro plasminogen, plasminogen activator, prolactin, proopiomelanocortin, protein C, prothrombin, relaxin, prepro renin, somatostatin, prepro tachykinin, substance P, substance K, urokinase and prepro vasoactive intestinal peptide protein.
Particular preferably proteins are GP63, myelin basic protein, hCD59, Factor IX, xcex1-antitrypsin, xcex1-casein, interleukins and Bcl-2.
The viable G0/G1 cells of the invention may further comprise one or two exogenous DNA segments, wherein the one or two exogenous DNA segments comprise at least a first and second coding region that each express a selected protein. Certain advantages are wherein the first coding region encodes a physiologically or pharmacologically active protein or RNA and the second coding region encodes a selected marker protein. In certain embodiments, such coding regions are preferably on the same exogenous DNA segment.
The exogenous DNA segment(s) may be operatively positioned under the control of an exogenous promoter that directed expression in the chosen cell type, although the use of an exogenous promoter is not necessary to the practice of the invention. The selected DNA segment(s) may be introduced into the cells by any suitable method, such as, e.g., by electroporation, particle bombardment, viral transformation or such like.
In certain preferred embodiments, the exogenous DNA segment further comprises two selected DNA regions that flank the DNA segment, thereby directing the homologous recombination of the DNA segment into the genomic DNA of the target cells, i.e., the viable G0/G1 cells. In such embodiments, the DNA segment may further comprise two selected DNA sequences that flank the DNA segment, thereby directing excision of the DNA segment under appropriate conditions. Examples of such selected DNA sequences are loxP sites, for use with the Cre Lox system. Such homologous recombination techniques are disclosed in U.S. application Ser. No. 08/949,155, incorporated herein by reference.
Also as disclosed in U.S. application Ser. No. 08/949,155, incorporated herein by reference, this invention therefore concerns methods of preparing mammalian cells at the G0/G1 stage of the cell cycle that contain a selected DNA segment, comprising (a) culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in media comprising an amount of at least a first apoptosis inhibitor effective to increase the proportion of viable G0/G1 cells in the cell population; and (b) introducing a selected DNA segment into the viable G0/G1 cells in said cell population.
The present invention is applicable to all animals, particularly valuable or valued animals, such as farm animals used to produce food for human consumption and breeding stock, race horses, domestic pets, zoological animals and research animals. In addition, for aspects concerning the generation of cells, not whole animals, the present invention applies to producing human cells, e.g., for culture and/or use in human treatment. Thus, xe2x80x9ctransgenic and cloned whole mammalsxe2x80x9d exclude humans.
In particular aspects of the invention, the mammalian cell populations therefore contain cells from a lagomorph (gnawing, herbivorous mammal, e.g., rabbit), bovine (cow), porcine (pig,), ovine (sheep), equine (horse), caprine (goat), canine (dog), feline (cat), murine (mouse), non-human primate (monkey, chimpanzee, etc.) or human primate species. Cells from boar, buffalo, bison, llama, deer, elk, lion, tiger, zebra, giraffe, elephant, panda, and other large animals, as well as their young, are also included. As nuclear transfer technology has been applied outside the field of mammals, cells from non-mammals, such as birds, amphibians and fish are included, particularly commercially relevant birds, such as chicken, turkey, duck, goose, ostrich, emus, dove, quail, and the like.
In certain embodiments, the apoptosis inhibitors for use in the invention will be one or more serine protease-type apoptosis inhibitors (xe2x80x9cserine protease inhibitorsxe2x80x9d) or antioxidant-type apoptosis inhibitors (xe2x80x9cantioxidantsxe2x80x9d). Preferred serine protease inhibitors include, but are not limited to, xcex12-macroglobulin (MAC), uteroferrin rose, 4-(2-aminoethyl) benzenesulfonyl hydrochloride (AEBSF), N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), 3,4-dichloroisocoumarin, serpins and E64 class serine protease inhibitors. MAC, AEBSF and TLCK are currently preferred. MAC is particularly preferred, with concentrations of between about 0.3 and about 1.50 pM MAC being more preferred, and about 0.7 pM MAC being particularly preferred.
Preferred antioxidants include, but are not limited to, N-acetylcysteine (NAC), butylated hydroxyanisole (BHA), cimetidine (CIM), N-t-butyl-xcex1-phenylnitrone (BPN), thioredoxin and glutathione (GSH). NAC, BHA, BPN, CIM and GSH are preferred, particularly at concentrations about those listed in Table 7 or about 2.0 mM for GSH. NAC is one such preferred agent, with concentrations of between about 0.2 and about 3.0 mM or more NAC being preferred, such as at least about 2.0 mM NAC or about 2.0 mM NAC being particularly preferred.
In certain preferred embodiments of the invention, at least a first and second apoptosis inhibitor is used. For example, distinct serine protease inhibitors, distinct antioxidants, or combinations of serine protease inhibitors and antioxidants, such as combinations of xcex12-macroglobulin and N-acetylcysteine. In further aspects of the invention, three, four, five, six, or more, such as a plurality of apoptosis inhibitors, are used.
In that the cell populations of the invention need to be cultured, a convenient method of obtaining a starting cell population of mammalian cells comprising potentially nuclear transfer competent cells, such as potentially viable G0/G1 cells, is to culture the cell population in serum starvation media. Accordingly, the serum starvation media will preferably contain the at least a first apoptosis inhibitor for use in the invention.
In certain embodiments, the serum starvation media will comprise between about 0.05% and about 2% serum. It will be understood that all sub-ranges are included within this range, such as between about 0.1% and about 2%, between about 0.25% and about 2%, between about 0.5% and about 2%, between about 1% and about 2%, between about 1.5% and about 2%, between about 0.05% and about 1.5%, between about 0.05% and about 1%, between about 0.05% and about 0.5%, between about 0.05% and about 0.25%, between about 0.05% and about 0.1%, between about 0.1% and about 1.5%, between about 0.25% and about 1% and between about 0.5% and about 0.75% and such like. In preferred embodiments, the serum starvation media comprises between about 0.1% and about 0.5% serum. In other aspects, the serum starvation media comprises about 0.05%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about 0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about 1.1%, about 1.2%, about 1.25%, about 1.3%, about 1.4%, about 1.5 %, about 1.6%, about 1.7%, about 1.75%, about 1.8% or about 1.9% serum and such like.
In certain aspects of the invention, the nuclear transfer competent or viable G0/G1 cell(s) and the enucleated recipient cell or ovum are from the same mammalian species, while in other aspects, the nuclear transfer competent or viable G0/G1 cell(s) and the enucleated recipient cell or ovum are from distinct mammalian species, for example a human cell and a bovine enucleated ovum, for the generation of tissues for transplantation into humans. In preferred aspects of the invention, the nuclear transfer competent or viable G0/G1 cell(s) and the enucleated recipient cell or ovum are from a lagomorph, bovine, porcine, ovine, equine, caprine, canine, feline, murine, non-human primate, or human primate species. Cells from boar, buffalo, bison, llama, deer, elk, lion, tiger, zebra, giraffe, elephant, panda, and other large animals, as well as their young, are also included.
In further aspects of the invention, a population of mammalian cells is cultured and a single viable G0/G1 cell from the population is fused with the enucleated mammalian ovum. Irrespective of the number of cells for fusion, the cells may be cultured under the conditions of the invention for between about 3 and about 30 days; preferably, for between about 5 and about 14 days; and more preferably, for about 10 days or so. With any and all intermediate and partial ranges being included, such as 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and the like.
An exemplary method of the invention comprises culturing a cell population containing bovine or porcine fetal or embryonic fibroblasts or primordial germ cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of at least a first apoptosis inhibitor effective to increase the proportion of viable G0/G1 cells in the cell population; and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated bovine or porcine ovum, thereby achieving nuclear transfer. Preferably, the apoptosis inhibitor is a MAC, NAC, BHA, CIM, BPN or GSH apoptosis inhibitor, and more preferably, the media comprises at least a first serine protease apoptosis inhibitor and at least a second antioxidant apoptosis inhibitor.
Another exemplary method of the invention comprises culturing a mammalian cell population, preferably a bovine or porcine fetal or embryonic fibroblast or primordial germ cell population, containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an amount of the apoptosis inhibitor xcex12-macroglobulin (MAC) effective to increase the proportion of viable G0/G1 cells in the cell population when present substantially throughout the culture of said cell population; and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated mammalian ovum, thereby achieving nuclear transfer.
A still further exemplary method of the invention comprises culturing a mammalian cell population, preferably a bovine or porcine fetal or embryonic fibroblast or primordial germ cell population, containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising an apoptosis inhibitor selected from the group consisting of N-acetylcysteine (NAC), butylated hydroxyanisole (BHA), cimetidine (CIM), N-t-butyl-xcex1-phenylnitrone (BPN) and glutathione (GSH) in an amount effective to increase the proportion of viable G0/G1 cells in the cell population; and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated mammalian ovum, thereby achieving nuclear transfer.
A yet further exemplary method of the invention comprises culturing a mammalian cell population, preferably a bovine or porcine fetal or embryonic fibroblast or primordial germ cell population, containing cells at the G0G1 stage of the cell cycle in serum starvation media comprising an amount of the apoptosis inhibitor N-acetylcysteine (NAC) effective to increase the proportion of viable G0/G1 cells in the cell population when present at the initial stages of the culture of said cell population; and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated mammalian ovum, thereby achieving nuclear transfer.
Yet another exemplary method of the invention comprises culturing a mammalian cell population, preferably a bovine or porcine fetal or embryonic fibroblast or primordial germ cell population, containing cells at the G0/G1 stage of the cell cycle in serum starvation media comprising at least a first serine protease apoptosis inhibitor and at least a second antioxidant apoptosis inhibitor in a combined effective to increase the proportion of viable G0/G1 cells in the cell population; and fusing at least a first viable G0/G1 cell from the cultured cell population with an enucleated bovine or porcine ovum, thereby achieving nuclear transfer.
In certain embodiments, the use of xcex12-macroglobulin is excluded from the invention, so that the invention concerns methods of nuclear transfer, and associated methods and compositions, all of which comprise culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in media comprising at least a first serine protease apoptosis inhibitor other than xcex12-macroglobulin in an amount effective to increase the proportion of viable G0/G1 cells in the cell population; and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum.
In certain other embodiments, the use of the antioxidant apoptosis inhibitor thioredoxin is specifically excluded from the invention, so that the invention concerns methods of nuclear transfer, and associated methods and compositions, all of which comprise culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in media comprising at least a first antioxidant apoptosis inhibitor other than thioredoxin in an amount effective to increase the proportion of viable G0/G1 cells in the cell population; and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum.
In yet other embodiments, the use of uteroferrin is specifically excluded from the invention, so that the invention concerns methods of nuclear transfer, and associated methods and compositions, all of which comprise culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in media comprising at least a first apoptosis inhibitor other than uteroferrin in an amount effective to increase the proportion of viable G0/G1 cells in the cell population; and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum.
In still further embodiments, the use of xcex12-macroglobulin, thioredoxin and uteroferrin is specifically excluded from the invention, so that the invention concerns methods of nuclear transfer, comprising culturing a mammalian cell population containing cells at the G0/G1 stage of the cell cycle in media comprising at least a first apoptosis inhibitor other than xcex12-macroglobulin, thioredoxin or uteroferrin in an amount effective to increase the proportion of viable G0/G1 cells in the cell population; and fusing at least a first viable G0/G1 cell with an enucleated mammalian ovum.
The present invention also provides methods of cloning a mammal from a somatic or germ cell from a mammalian adult, fetus or embryo, comprising (a) culturing a population of mammalian somatic or germ cells containing cells at the G0/G1 stage of the cell cycle in media comprising an effective amount of at least a first apoptosis inhibitor for a period of time suitable to increase the proportion of viable G0/G1 cells in the population; and (b) generating a viable cloned mammal from at least a first of the viable G0/G1 cells.
This invention further provides methods of producing transgenic mammals, comprising (a) culturing a population of mammalian somatic or germ cells containing cells at the G0/G1 stage of the cell cycle in media comprising an effective amount of at least a first apoptosis inhibitor for a period of time suitable to increase the proportion of viable G0/G1 cells in the population; (b) introducing a selected DNA segment into viable G0/G1 cells of the cell population to produce viable transgenic G0/G1 cells; and (c) generating a transgenic animal from at least a first of the viable transgenic G0/G1 cells, wherein the selected DNA segment is contained and expressed in somatic and germ cells of the transgenic animal.
Whether xe2x80x9cclonedxe2x80x9d or xe2x80x9ctransgenicxe2x80x9d, various methods are available to xe2x80x9cgeneratexe2x80x9d the mammals from at least a first of the viable, optionally transgenic G0/G1 cells. For example, methods involving preparing blastocysts, preparing oocytes and preparing early stage embryos are included.
One may thus (a) fuse at least a first of the viable, optionally transgenic G0/G1 cells with an enucleated mammalian ovum (oocyte); (b) transferring the fused cell/ovum into a synchronized recipient mammalian female to produce a pregnant mammal; and (c) allowing gestation in the pregnant mammal to proceed for a period of time effective to allow the development of a viable cloned or transgenic mammal. Similarly, within step (a), one could (i) isolate a nucleus from the viable, optionally transgenic G0/G1 cells and (ii) inject the nucleus into an enucleated mammalian ovum (oocyte); and then continue with steps (b) and (c) as above.
Equally, the viable, optionally transgenic G0/G1 cells may be (a) injected into a blastocyst from a suitable mammal; followed by (b) transferring the blastocyst into a synchronized recipient female mammal to produce a pregnant mammal; and (c) allowing gestation in the pregnant mammal to proceed for a period of time sufficient to allow the development of a viable cloned or transgenic mammal.
Further, the viable, optionally transgenic G0/G1 cells may be (a) aggregated with an early stage embryo of a suitable mammal; followed by (b) transferring the embryo into a synchronized recipient female mammal to produce a pregnant mammal; and (c) allowing gestation in the pregnant mammal to proceed for a period of time sufficient to allow the development of a viable cloned or transgenic mammal.
The present invention further provides methods of producing a chimeric mammal from a somatic mammalian cell, comprising (a) culturing a population of somatic mammalian cells containing cells at the G0/G1 stage of the cell cycle in media comprising an effective amount of at least a first apoptosis inhibitor for a period of time suitable to increase the proportion of viable G0/G1 somatic cells in the population; (b) fusing at least a first of the viable G0/G1 somatic cells with an enucleated mammalian ovum; (c) culturing the fused cell/ovum in embryo media for a period of time effective to reach the morula/blastocyst stage of development; (d) combining the morula/blastocyst with a morula/blastocyst from a distinct mammalian species to form a morula/blastocyst aggregate; (e) transferring the morula/blastocyst aggregate into a synchronized recipient mammalian female to produce a pregnant mammal; and (f) allowing gestation in the pregnant mammal to proceed for a period of time effective to allow the development of a viable chimeric mammal.
Additionally, the present invention provides methods of producing mammalian cell lines. Using the present invention in conjunction with U.S. application Ser. No. 08/949,155, incorporated herein by reference, provides methods of preparing mammalian cell lines from somatic or germ cells, comprising (a) culturing a cell population comprising mammalian somatic or germ cells on an effective density of feeder cells and in a biologically effective culture medium comprising an amount of at least a first apoptosis inhibitor effective to increase the number of nuclear transfer competent cells in the cell population during culture; and (b) maintaining the cultured cell population for a period of time effective to provide a mammalian cell line.
The cell line methods of the present invention further include methods of producing a mammalian cell line from a somatic mammalian cell, comprising (a) culturing a population of mammalian somatic cells containing cells at the G0/G1 stage of the cell cycle in media comprising an effective amount of at least a first apoptosis inhibitor for a period of time suitable to increase the proportion of viable G0/G1 somatic cells in said population; (b) fusing at least a first of the viable G0/G1 somatic cells with an enucleated mammalian ovum; (c) culturing the fused cell/ovum in suitable media, such as embryo media, for a period of time effective to reach the morulalblastocyst stage of development, and (d) culturing the morula/blastocyst in suitable media, such as complete media, with or without a feeder layer and/or growth factors, for a period of time effective to allow the development of a mammalian cell line.
Yet further aspects of the invention include compositions, cell cultures and/or kits comprising cell culture media that comprise an amount of at least a first apoptosis inhibitor effective to increase the proportion of viable G0/G1 cells in a mammalian cell population and instructions for using the cell culture media to increase the proportion of viable G0/G1 cells in a mammalian cell population when cultured using the compositions, cell cultures and/or kits.
Other aspects of the invention are compositions, cell cultures and/or kits comprising a mammalian cell population containing cells at the G0/G1 stage of the cell cycle and a cell culture media that comprise an amount of at least a first apoptosis inhibitor effective to increase the proportion of viable G0/G1 cells in a mammalian cell population. Instructions for using the cell culture media to increase the proportion of viable G0/G1 cells in the mammalian cell population may be included in such compositions, cell cultures and/or kits.
The entire range of apoptosis inhibitors and combinations thereof, as exemplified herein in terms of the methods of the invention, may be used in the compositions and kits of the invention. In all such compositions, cell cultures and/or kits, further components may be included, such as DNA segments, vectors, feeder cells, various container and apparatus for confining the components. U.S. application Ser. No. 08/949,155 is specifically incorporated herein by reference for purposes of further describing such compositions, cell cultures and/or kits and their combination with other biological components and apparatus.